Preparation and Identification of Recombinant Sarcosine Oxidase_Journal of Biomedical Engineering

Authors：

PUJing ¹ , WANGRui ² , YAOMingdong ² , HEZhongjie ² , ZHAOMing ² ,  MENGYao ²

1. The First Affiliated Hospital of Chengdu Medical College, Chengdu 610083, China;
2. School of Medical Laboratory Science, Chengdu Medical College, Chengdu 610500, China;

Corresponding author：

MENGYao, Email: myaopapers@gmail.com

Keywords：

recombinant sarcosine oxidase; inducible expression; high density fermentation

DOI：

10.7507/1001-5515.20140205

Video：

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An important index determination for clinical diagnosis of renal function is to assay the creatinine concentration in serum. In the analytical process applied with coupled-enzyme, the quality control of sarcosine oxidase (SOX) as a key enzyme is the first problem to be solved. In order to establish an efficient and laboratory-scale production of SOX, the recombinant sarcosine oxidase (r-SOX) gene was a high-level expression in E.coli induced with lactose on a large-scale fermentation in 300 L fermenter. The results suggested that the biomass concentration reached OD₆₀₀ of 22 and the expression of recombinant sarcosine oxidase in E. coli accounted for about 25% of total soluble protein in culture after fermentation. The cell-free extract obtained from high pressure homogenizer was processed by selective thermal denaturation and then purified with Ni-Sepharose FF chromatography. The sarcosine oxidase with 97% purity, 25 U/mg specific activity and 92.4% activity recovery was obtained. The molecular weight with single peptide chain of 53 kD and 55 kD of recombinant sarcosine oxidase was assessed by SDS-PAGE in presence or absence of 2-mercaptoehanol and Sephacryl S-200 chromatography. This sarcosine oxidase was found to be a conjugated protein, yellow enzyme, which combined with FAD as prosthetic group by covalent linkage. The contaminant of catalase was not detected in the sample pool of this enzyme. In addition, a further test to the thermal stability of sarcosine oxidase was done. According to the above results, the development and utilization of this enzyme has been set up on a reliable foundation.

Citation： PUJing, WANGRui, YAOMingdong, HEZhongjie, ZHAOMing, MENGYao. Preparation and Identification of Recombinant Sarcosine Oxidase. Journal of Biomedical Engineering, 2014, 31(5): 1090-1096. doi: 10.7507/1001-5515.20140205 Copy

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2.	TOMBACH B, SCHNEIDER J, MATZKIES F, et al. Amperometric creatinine biosensor for hemodialysis patients[J]. Clin Chim Acta, 2001, 312(1-2): 129-134.
3.	SUZUKI M. Purification and some properties of sarcosin oxidase from Corynebacterium sp. U-96[J]. J Biochem, 1981,89(2):599-607.
4.	BACON B L, PARDUE H L. Predictive, error-compensating kinetic method for enzymatic quantification of creatinine in serum[J]. Clin Chem, 1991, 37(8): 1338-1344.
5.	KINOSHITA T, HIRAGA Y. A fluorophotometric determination of serum creatinine and creatine using a creatinineamidohydrolase-creatineamidinohydrolase-sarcosine oxidase-peroxidase system and diacetyldichlorofluorescin[J]. Chem Pharm Bull (Tokyo), 1980, 28(12): 3501-3506.
6.	LINDBÄCK B, BERGMAN A. A new commercial method for the enzymatic determination of creatinine in serum and urine evaluated: comparison with a kinetic Jaffé method and isotope dilution-mass spectrometry[J]. Clin Chem, 1989, 35(5): 835-837.
7.	SUZUKI H. Sarcosine oxidase: structure, function, and the application to creatinine determination[J]. Amino Acids, 1994, 7(1): 27-43.
8.	OKUMIYA T, JIAO Y, SAIBARA T, et al. Sensitive enzymatic assay for erythrocyte creatine with production of methylene blue[J]. Clin Chem, 1998, 44(7): 1489-1496.
9.	CHLUMSKY L J, ZHANG L, RAMSEY A J, et al. Preparation and properties of recombinant corynebacterial sarcosine oxidase: evidence for posttranslational modification during turnover with sarcosine[J]. Biochemistry, 1993, 32(41): 11132-11142.
10.	MATSUDA Y, HOSHIKA H, INOUYE Y, et al. Purification and characterization of sarcosine oxidase of Bacillus origin[J]. Chem Pharm Bull (Tokyo), 1987, 35(2): 711-717.
11.	NISHIYA Y, IMANAKA T. Cloning and sequencing of the sarcosine oxidase gene from Arthrobacter sp.TE1826[J]. J Ferment Bioeng,1993,75(4):239-244.
12.	SUZUKI K, OGISHIMA M, SUGIYAMA M, et al. Molecular cloning and expression of a Streptomyces sarcosine oxidase gene in Streptomyces lividans[J]. Biosci Biotechnol Biochem, 1992, 56(3): 432-436.
13.	SUZUKI K, SAGAI H, IMAMURA S, et al. Cloning, sequencing and overexpression in Escherichia coli of a sarcosine oxidase-encoding gene linked to the Bacillus creatinase gene[J]. J Ferment Bioeng,1994,77(3):231-234.
14.	RIESENBERG D, GUTHKE R. High-cell-density cultivation of microorganisms[J]. Appl Microbiol Biotechnol, 1999, 51(4): 422-430.
15.	张毅,屈贤铭,杨胜利.乳糖作为诱导剂对重组目的蛋白表达的影响[J].生物工程学报,2000,16(4):464-468.
16.	李兆鹏,张栩,徐斌,等.重组大肠杆菌高密度发酵中乳糖诱导表达hBLyS[J].过程工程学报,2005,5(4):446-449.
17.	BRADFORD M M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[J]. Anal Biochem, 1976, 72: 248-254.
18.	Toyobo Co., Ltd., Biochemical Dept. Toyobo enzymes[M]. Japan: Academic Press, 1996: 112-115.
19.	GUO K P, MA X H, SUN G Q, et al. Expression and characterization of a thermostable sarcosine oxidase (SOX) from Bacillus sp. in Escherichia coli[J]. Appl Microbiol Biotechnol, 2006, 73(3):559-566.
20.	LEMMLI U K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4[J]. Nature, 1970, 227(5259): 680-685.
21.	MARSHAK D R, KADONAGA J T, BURGESS R R, et al. Strategies for protein purification and characterization: A laboratory course manual[M]. New York: Cold Spring Harbor Laboratory Press, 1996.
22.	DEUTSCHER M P. Maintaining protein stability[J]. Methods Enzymol, 1990, 182: 83-89.
23.	ANDREWS A T. Electrophoresis: theory, techniques and biochemical and clinical applications[M]. Oxford and New York: Clarendon Press, 1986.
24.	ARGOS P, ROSSMAN M G, GRAU U M, et al. Thermal stability and protein structure[J]. Biochemistry, 1979, 18(25):5698-5703.
25.	KOYAMA Y, YAMAMOTO-OTAKE H, SUZUKI M, et al. Cloning and expression of the sarcosine oxidase gene from Bacillus sp. NS-129 in Escherichia coli[J]. Agric Biol Chem, 1991, 55(5): 1259-1263.
26.	TRICKEY P, WAGNER M A, JORNS M S, et al. Monomeric sarcosine oxidase: structure of a covalently flavinylated amine oxidizing enzyme[J]. Structure, 1999, 7(3): 331-345.

1. ERLENKÖTTER A, FOBKER M, CHEMNITIUS G C. Biosensors and flow-through system for the determination of creatinine in hemodialysate[J]. Anal Bioanal Chem, 2002, 372(2): 284-292.
2. TOMBACH B, SCHNEIDER J, MATZKIES F, et al. Amperometric creatinine biosensor for hemodialysis patients[J]. Clin Chim Acta, 2001, 312(1-2): 129-134.
3. SUZUKI M. Purification and some properties of sarcosin oxidase from Corynebacterium sp. U-96[J]. J Biochem, 1981,89(2):599-607.
4. BACON B L, PARDUE H L. Predictive, error-compensating kinetic method for enzymatic quantification of creatinine in serum[J]. Clin Chem, 1991, 37(8): 1338-1344.
5. KINOSHITA T, HIRAGA Y. A fluorophotometric determination of serum creatinine and creatine using a creatinineamidohydrolase-creatineamidinohydrolase-sarcosine oxidase-peroxidase system and diacetyldichlorofluorescin[J]. Chem Pharm Bull (Tokyo), 1980, 28(12): 3501-3506.
6. LINDBÄCK B, BERGMAN A. A new commercial method for the enzymatic determination of creatinine in serum and urine evaluated: comparison with a kinetic Jaffé method and isotope dilution-mass spectrometry[J]. Clin Chem, 1989, 35(5): 835-837.
7. SUZUKI H. Sarcosine oxidase: structure, function, and the application to creatinine determination[J]. Amino Acids, 1994, 7(1): 27-43.
8. OKUMIYA T, JIAO Y, SAIBARA T, et al. Sensitive enzymatic assay for erythrocyte creatine with production of methylene blue[J]. Clin Chem, 1998, 44(7): 1489-1496.
9. CHLUMSKY L J, ZHANG L, RAMSEY A J, et al. Preparation and properties of recombinant corynebacterial sarcosine oxidase: evidence for posttranslational modification during turnover with sarcosine[J]. Biochemistry, 1993, 32(41): 11132-11142.
10. MATSUDA Y, HOSHIKA H, INOUYE Y, et al. Purification and characterization of sarcosine oxidase of Bacillus origin[J]. Chem Pharm Bull (Tokyo), 1987, 35(2): 711-717.
11. NISHIYA Y, IMANAKA T. Cloning and sequencing of the sarcosine oxidase gene from Arthrobacter sp.TE1826[J]. J Ferment Bioeng,1993,75(4):239-244.
12. SUZUKI K, OGISHIMA M, SUGIYAMA M, et al. Molecular cloning and expression of a Streptomyces sarcosine oxidase gene in Streptomyces lividans[J]. Biosci Biotechnol Biochem, 1992, 56(3): 432-436.
13. SUZUKI K, SAGAI H, IMAMURA S, et al. Cloning, sequencing and overexpression in Escherichia coli of a sarcosine oxidase-encoding gene linked to the Bacillus creatinase gene[J]. J Ferment Bioeng,1994,77(3):231-234.
14. RIESENBERG D, GUTHKE R. High-cell-density cultivation of microorganisms[J]. Appl Microbiol Biotechnol, 1999, 51(4): 422-430.
15. 张毅,屈贤铭,杨胜利.乳糖作为诱导剂对重组目的蛋白表达的影响[J].生物工程学报,2000,16(4):464-468.
16. 李兆鹏,张栩,徐斌,等.重组大肠杆菌高密度发酵中乳糖诱导表达hBLyS[J].过程工程学报,2005,5(4):446-449.
17. BRADFORD M M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[J]. Anal Biochem, 1976, 72: 248-254.
18. Toyobo Co., Ltd., Biochemical Dept. Toyobo enzymes[M]. Japan: Academic Press, 1996: 112-115.
19. GUO K P, MA X H, SUN G Q, et al. Expression and characterization of a thermostable sarcosine oxidase (SOX) from Bacillus sp. in Escherichia coli[J]. Appl Microbiol Biotechnol, 2006, 73(3):559-566.
20. LEMMLI U K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4[J]. Nature, 1970, 227(5259): 680-685.
21. MARSHAK D R, KADONAGA J T, BURGESS R R, et al. Strategies for protein purification and characterization: A laboratory course manual[M]. New York: Cold Spring Harbor Laboratory Press, 1996.
22. DEUTSCHER M P. Maintaining protein stability[J]. Methods Enzymol, 1990, 182: 83-89.
23. ANDREWS A T. Electrophoresis: theory, techniques and biochemical and clinical applications[M]. Oxford and New York: Clarendon Press, 1986.
24. ARGOS P, ROSSMAN M G, GRAU U M, et al. Thermal stability and protein structure[J]. Biochemistry, 1979, 18(25):5698-5703.
25. KOYAMA Y, YAMAMOTO-OTAKE H, SUZUKI M, et al. Cloning and expression of the sarcosine oxidase gene from Bacillus sp. NS-129 in Escherichia coli[J]. Agric Biol Chem, 1991, 55(5): 1259-1263.
26. TRICKEY P, WAGNER M A, JORNS M S, et al. Monomeric sarcosine oxidase: structure of a covalently flavinylated amine oxidizing enzyme[J]. Structure, 1999, 7(3): 331-345.

Journal of Biomedical Engineering

Preparation and Identification of Recombinant Sarcosine Oxidase

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